1. Field of the Invention
The present invention relates generally to the fields of pest control and molecular biology and, in particular, to genetic control of programmed cell death (PCD), or apoptosis in insect and human cells.
2. Description of Related Art
Programmed cell death (PCD) or apoptosis is an essential regulator of tissue differentiation and cellular maintenance as animals develop and age [Saunders and Fallon, 1967; Truman, 1984; Hurle, 1988; Ellis, et al., 1991; Oppenheim, 1991; Raff, 1992]. Recent studies have implicated the misregulation of apoptosis in the pathophysiology of several human diseases including AIDS [Meyaard, et al., 1992; Gougen and Montagnier, 1993], neurodegenerative disease [Roy, et al., 1995; Liston, et al., 1996; Vito, et al., 1996] and cancer [reviewed in Williams, 1991].
Several lines of evidence suggest that the physiology of apoptosis is quite highly conserved. First, the morphological changes associated with programmed cell'deaths are stiringly similar in both vertebrates and invertebrates [Kerr, et al., 1972; Wyllie, et al., 1980; Kerr and Harmon, 1991; Abrams, et al, 1993]. Second, at least two essential cell death genes in Caenorhabditis elegans, ced-3 and ced-9, are members of gene families that encode apoptotic functions in vertebrates [reviewed in Steller, 1995; White, 1996). Third, viral proteins that suppress apoptosis in their hosts (p35 and, crmA) can exhibit potent anti-apoptotic activity in a wide range of heterologous species Rabizadeh, et al., 1993; Hay, et al., 1994; Sugimoto, et al., 1994; Grether, et al, 1995; Pronk, et al., 1996; White, et al., 1996].
In Drosophila melanogaster, a genomic interval defined by the H99 deletion mutation is required for embryonic programmed cell death [Abrams, et al., 1993; White, et al., 1994; Abrams, 1996]. This region spans .about.300 kilobases of DNA that includes at least two cell death genes, reaper (rpr) [White, et al., 1994] and head involution defective. (hid) [Grether, et al., 1995]. The former gene product is thought to share similarities to the "death domain" of the FAS/TNFR1 protein family [Cleveland and Ihle, 1995; Golstein, et al., 1995; Golstein, et al., 1995] whereas the latter shares no extensive sequence similarity to known proteins. Although the distribution of RNA from both genes generally corresponds to embryonic patterns of apoptosis, only rpr appears to be selectively expressed in all cells that will later undergo programmed cell death.
Although no apoptosis occurs in embryos bearing homozygous deletions of the entire H99 interval [Abrams, et al., 1993; White, et al., 1994], null mutations at hid display only mild cell death defects [Grether, et al., 1995] and, to date, no single-gene mutants of the rpr have been identified. Therefore, the precise number of cell death genes uncovered by H99 is not known. In fact, phenotypes associated with two informative deletions in the region, X14 and X25 [White, et al., 1994; Grether, et al., 1995], raised the possibility that perhaps one or more additional cell death genes might reside between hid and rpr. Both strains partially uncover the H99 interval from the distal boundary thereby eliminating hid yet preserving rpr. However, although both deletions exhibit mild and indistinguishable PCD phenotypes as homozygotes [Grether, et al., 1995], X25 uncovers a far more severe phenotype when placed in trans to H99 than does X14. Whereas X14/H99 embryos show subtle cell death defects similar to those observed for hid null alleles, X25/H99 transheterozygotes exhibit a severe reduction in apoptosis frequency that can be easily visualized by staining with acridine orange [Grether, 1994]. Since the relevant breakpoint of X25 is .about.60kb more proximal than that of X14 [Grether, 1994], it is possible that one or more additional cell death functions map to the interval bounded by these breakpoints.
There is still a need therefore, for methods of controlling insect pests without the use of pesticides that remain in the environment and contribute to pesticide resistance, or for methods of controlling apoptosis in cells that have a disrupted apoptotic function by restoration of apoptosis genes. These needs may be met by the discovery of apoptosis controlling genes that are active in insect species and that also may be active in other species, including mammalian and even human cells.